Biomimetic Scaffolds for Regeneration of Temporomandibular Joint Disc: A Narrative Review

Defects and dysfunctions of temporomandibular joint (TMJ) disc are responsible for the majority of TMJ diseases. Current treatments in this matter are usually short-term and only palliative, thus an alternative treatment that offers long-lasting repair is in great demand. In recent years great attempts have been made to prepare an ideal scaffold, which best resembles the native TMJ disc in characteristics such as mechanical, physical and biological properties. This narrative review focuses on developments of the recent ten years in fabrication of scaffolds using decellularized tissues, natural and synthetic biomaterials for regeneration of TMJ disc and compared their properties. PubMed and Google Scholar databases were searched using the following keywords (“TMJ” OR “temporomandibular joint” OR “TMD” OR “temporomandibular disease”) AND (“scaffold” OR “hydrogels”). Randomized controlled trials, randomized clinical trials, case-controls, case reports, and animal studies were included. Comments, systematic reviews, meta-analyses, and non-English papers were excluded. The study concluded that hybrid scaffolds have exhibited favorable cell attachment and proliferation. Synthetic scaffolds have shown promise in providing better control over structural properties; however, additional processes are often required to provide biomimetic cell signaling. While there is still much to learn about the ideal scaffold for TMJ disc regeneration, both natural and synthetic scaffolds have shown promise in achieving the functional, structural, biological, and mechanical properties of a native TMJ disc.


Introduction
Temporomandibular joint disease (TMD) excruciatingly reduces the comfort and quality of life of many people worldwide [1].TMD occurs as a result of dysfunctions or defects in the temporomandibular joint (TMJ) disc such as disc displacement which is believed to increase the possibility of perforation [2].The importance of TMJ disc is that it acts as a stress absorber and a distributor of stress [3].Disc may not recover from a perforation due to avascular nature, which eventually leads to impaired function [2].Several surgical options are suggested to relieve the pain such as discectomy; however, they are only anodynes, which usually require revision and come with significant post-surgical complications [4][5].Thus, tissue engineering has been studied as an alternative option for a rather long time and attempts have been made to prepare a scaffold that best resembles the features of native TMJ disc (Figure 1).
The native TMJ disc is a fibrocartilage, which mostly consists of water, as well as, cells and extracellular matrix (ECM) [6].Cellular and extracellular elements of TMJ disc have shown region-dependent distribution [5][6], which is considered to be responsible for viscoelastic and anisotropic tensile properties [7][8].Approximately 70% of cellular element is composed of fibroblast-like cells and the rest is composed of chondrocyte-like cells.
The former is found all over the tissue, while the latter is mostly found in the intermediate area [9].The  (GAG).GAGs are mainly localized in the intermediate area [5].
Aside from diverse composition and fiber orientation in different parts of the disc, mechanical properties (to withstand compressive, tensile and shear forces), biological properties (suitable for cell proliferation and differentiation), and physical properties (to provide mass transport) are all the challenges in regeneration of TMJ disc [6,[9][10].
In this narrative review we aimed to study materials that have already been fabricated and examined (wheth-er in vitro or in vivo) to prepare an inhomogeneous tissue capable of replacing TMJ disc.

Methods and search strategy
PubMed and Google Scholar databases were analyzed with the following keywords: ("TMJ" OR "temporomandibular joint" OR "TMD" OR "temporomandibular disease") AND ("scaffold" OR "hydrogels")

Inclusion and exclusion criteria
Randomized controlled trials, randomized clinical trials, case-controls, case reports, animal studies from the last 10 years (since 2013) were included.Comments, systematic review and meta-analyses and papers that were not written in English were excluded.

Discussion
Scaffolds are defined as support structures for tissue formation [11].Natural biomaterials, synthetic biomaterials, and decellularized tissues have widely been used as scaffolds in TMJ disc regeneration [12][13].Different materials and modifications have been investigated to achieve the best scaffold that can mimic functional, structural, biological, and mechanical properties of a native TMJ disc.Table 1 presents the included in vitro studies; Table 2 presents the included in vivo studies, and the scaffolds that have been used with their results.

Natural Biomaterials
Despite the emergence of synthetic scaffolds, application of natural scaffolds has not obsoleted yet.This is mostly due to the hydrophobicity of synthetic scaffolds, which is believed to restrict cell attachment, a process that fundamentally affects cell signaling, migration and proliferation .Protein-based scaffolds such as fibrin and collagen provide binding site for cell adhesion, while polysaccharide-based scaffolds such as alginate and chitosan require modification [16].

Collagen
Collagen as a natural, biodegradable, and biocompatible material with high tensile strength has attracted a lot of attention in tissue engineering and been used in several forms including sponges [17].Collagen sponge that is seeded with bone marrow stem cells is believed to completely repair a perforation of 1.6mm diameter in the center of the disc after 2 weeks [2].A perforation in this size did not seem to be able to repair completely even after 2 months without either the scaffold or stem cells [2].

Hyaluronic acid
Hyaluronic acid (HA) is one of the components of ECM whose scaffolds are famous for hydrophilicity and ca- pability of chemical modification [16].Addition of Thiolated hyaluronic acid polymer to collagen to fabricate an injectable hydrogel (HA-Collagen1) diminishes the degradation time and shrinkage of collagen hydrogel; however, it decreases the storage modulus and loss modulus, which means that the ability of the scaffold to either store energy elastically or dissipate stress decreases [18].It also illustrates favorable compressive modulus, cell spreading, cell adhesion, and cell proliferation [18].Biphasic calcium phosphate can be added to HA-Collagen1 to mimic the bone layer, while HA-Coll-agen1 mimics cartilage layer [18].The combination still maintains its biocompatibility [18].While comparing seeding either bone marrow stem cells or chondrocytes in HA-Collagen1-biphasic calcium phosphate hydrogel, the production of GAG is higher with the latter; howev er, production of aggrecan is higher with the former [18].
HA-Collagen1-biphasic calcium phosphate hydrogel seeded with both bone marrow stem cells and chondrocytes fully repairs a defect with 2mm diameter and 3 mm depth after 6 weeks.The regenerated tissue integrates well with the adjacent native tissue and have a homogenous distribution of collagen type 1 and 2 and same cell pattern as the native disc (parallel fibroblastlike cell in the superficial zone and columnar spherical cells in the deep zone).Absence of the cells results in an uneven reparative tissue, without collagen type 2 [18].

Chitosan and Alginate
Chitosan and Alginate are natural, biocompatible, and biodegradable polymers [19][20].Considering hydrophilicity of alginate and bioactivity of chitosan, a hybrid scaffold of these two polymers seeded with stem cells exhibits favorable cell attachment, proliferation, and chondrogenic differentiation [21].Besides, collagen type 2, unlike collagen type 1, is not expressed at all.[21].A noteworthy point is that addition of glutaraldehyde while preparing the chitosan-alginate scaffold is reported to increase distribution of cells inside the scaffold [21].The even distribution, unlike dense cellular clusters, increases cell development and proliferation [22].In terms of mechanical characteristics, the value of storage modulus of chitosan-alginate scaffold was same as the values of the peripheral area of a native TMJ disc and lower than the values of the central region [21].

Fibrin
Fibrin gels mimic the coagulation process, and forms a fibrin clot, which attaches to the tissue and provide a cell adhesion site [23].Addition of fibrin gel to fabricate a hybrid chitosan/fibrin scaffold increases cell seeding efficiency, cell spreading, biocompatibility, production of GAG content, expression of collagen type 1 compared to chitosan scaffold [24].Although without cells, no sign of repair is observed in vivo [24].

Synthetic Biomaterials
Despite all felicitous properties of natural scaffolds, they offer limited control over structural properties such as porosity, fiber diameter, and so on [25].Although synthetic scaffolds have overcome this issue, they usually require additional processes to provide biomimetic cell signaling [25].

Polycaprolactone
Polycaprolactone (PCL) is a biocompatible synthetic polymer with sufficient mechanical properties [26].The stiffness of a PCL scaffold is even rather higher than the native disc, which can facilitate placement of the scaffold [6].The size, density, and direction of PCL fibers in a scaffold are matters of importance in determining mechanical properties, as well as temperature during production [5][6].Filament enlargement from 200 µm to 300 µm would almost double the compressive modulus, which is probably related to a decrease in size of the pores; however, it decreases the yield stress [26].The PCL fibers with a size of 300 µm and twice parallel fibers to the alignment direction than the perpendicular ones with biomimetic fiber alignment (the alignment that resembles collagen fibers' alignment in peripheral and intermediate area), have shown a tensile modulus approximate to the TMJ disc's [5].Cross-arranged fi-bers of PCL endure forces from various directions and seem to be responsive to mechanical needs [27].A PCL /PLA (polylactide) 2D nano-membrane in which fiber alignment evokes region-dependent collagen alignment in native disc, has shown a higher compressive modulus, relaxation modulus, and tensile strength compared to a membrane with random fiber alignment [28].Direction of scaffold fibers not only determines mechanical properties but also influences the healing process and guides the arrangement of GAG content [28].PCL/PLA nano-membrane with random fiber alignment could not completely repair a native TMJ disc that had gone through 80% subtotal discectomy after 16 weeks, whereas the same membrane with biomimetic fiber alignment could repair it thoroughly [28].Temperature affects the mechanical properties by influencing porosity and scaffold's geometry [6].Higher temperature during fabrication, lessens the porosity, flattens the fibers, and enhances the possibility of change in the scaffolds structure [6].A study suggested that a temperature rise from 80ºC to 86ºC decreases the compressive modulus about 8 MPa and [6]; however, another study suggested that an increase from 78ºC to 80ºC increases the compressive modulus and yield stress [26].Since fibrocartilage regeneration time is rather long, slow degradation rate of a scaffold would benefit the regeneration process [29].PCL polymer seems to have a long degradation time of 2 to 4 years, which is longer than desired [26,29].Addition of poly lactic-co-glycolic acid microspheres is believed to accelerate its degradation time to a more suitable time [30].
Connective tissue growth factor as a factor contributed to fibroblastic differentiation and formation of fibrous matrix and transforming growth factor-β3 (TGF-β3) as a factor contributed to chondrogenic differentiation and formation of cartilaginous matrix has been added to a stem cell-seeded PCL scaffold via poly lacticco-glycolic acid microspheres [5].The former was added to the peripheral area and the latter to the intermediate area, in attempt to mimic native TMJ disc's ECM matrix [5].Addition of connective tissue growth factor and TGF-β3 not only resulted in a denser collagen type 1 matrix but also induced collage type 2 formation in the intermediate zone, whereas no collagen type 2 was found in their absence [5,30].Besides, it increased the coefficient of viscosity in both intermediate and periph-eral areas [5].It turned out that higher concentration of mentioned growth factors could cause a denser cartilaginous matrix (collagen type 2 and aggrecan) and a lower compressive modulus (closer to that of native disc).The former consequently alters viscoelastic properties of the scaffold [31], enhances coefficient of viscosity and diminishes ratio of relaxation modulus to instantaneous modulus (make them closer to that of native disc) [5].
The mentioned scaffold presented a constant release of connective tissue growth factor and TGF-β3 up to 42 days [5].In another study, addition of bone morphogenic protein-2 as a factor contributed to mineralization and collagen formation along with connective tissue growth factor and TGF-β3 to a stem cellseeded PCL scaffold was investigated [30].It presented a constant release of the factors up to 42 days, complete in vivo degradation and tissue replacement after 4 weeks, region-dependent ECM matrix same as native disc and collagen rich matrix whose fibers' alignments were same as native disc [30].

Poly (ethylene glycol) diacrylate
Application of hybrid constructs such as poly (ethylene glycol) diacrylate (PEGDA) hydrogels along with PCL scaffolds are believed to have great potential [32].The reasoning is that PCL scaffold can provide proper mechanical properties, while the hydrogel filling the pores can provide proper cell and growth factor distribution and attachment [6,32].Attempts have been made to improve mechanical properties of PEGDA hydrogel in combination with PCL scaffold.Increase in PEGDA concentration in hydrogel from 20% to 30%, doubles the compressive modulus and triples the ultimate strength; however, the hydrogel with 30% concentration has turned out to be highly brittle [6].Increase in concentration also reduces the degradation rate [26].Addition of PEGDA hydrogel in form of a core and a shell to a PCL scaffold has been studied [26].The hydrogel core keeps the integrity of the structure, withstands large forces for a long period of time, and mimics mechanical properties of native disc; however, the hydrogel shell mimics the surface properties of native such as storing and diffusing synovial fluid, reducing friction, and reducing stress concentration by spreading forces [26].
The natural hydrophobicity of PCL scaffold have been tried to be diminished by NaOH solution surface treatment [6].Although, NaOH solution improves hy-drophilicity and thus cellular attachment, it decreases mechanical properties of PCL scaffold [6].
Carbon nanotubes are promising reinforcements due to mechanical strength and chemical stability [33].Addition of carbon nanotubes to a PCL/PLA Nanomembrane up to 0.25 wt% could increase the tensile strength.However, region-dependent fiber alignment has a greater impact on compressive and relaxation modulus than addition of 0.25 wt% carbon nanotubes, and application of both has a synergistic effect [28].
Carbon nanotubes could also increase hydrophilicity, biocompatibility, cell distribution, cell proliferation, and fibro-chondrogenic differentiation [34,35].A PCL/PLA nano-membrane with biomimetic fiber alignment and 0.25 wt% carbon nanotubes is capable of regenerating an anisotropic tissue with well-organized collagen fibers and GAG distribution same as native disc which majorly accumulates in the intermediate area and displays limited expression in the peripheral area [28].
PLA is a synthetic polymer with rather long degradation time, which makes it an option for tissue regeneration [36].Addition of transforming growth factor β1 to a PLA scaffold seeded with adipose stem cells increase production of aggrecan and collagen type 1; however, the scaffold completely repairs a TMJ with 90% subtotal discectomy after 6 months whether with or without transforming growth factor β1 [36].Although, the addition of transforming growth factor β1 results in a smoother tissue, which is more calcified near the condylar bone [36].

Polyvinyl alcohol
Polyvinyl alcohol (PVA) is a hydrophilic synthetic polymer [37].PVA in hydrogel form can provide lubrication by maintaining a substantial amount of water; however, it lacks suitable mechanical strength [27,38].Recently, in an attempt to improve mechanical properties, addition of PCL implants into the PVA hydrogel has been studied [27].PCL implants increase Young's modulus of PVA hydrogel.PVA hydrogel+ PCL implant also illustrates sufficient tensile strength to withstand functional forces, less creep than native disc, proper viscoelastic properties, and it recovers initial maximum strength after cyclic compressive stress even better than the native TMJ disc of a goat, which means that it could absorb destructive energy [27].Another problem with PVA, as well as PVA hydrogel+ PCL implant is that although they are biocompatible, their surface is not favorable for cell adhesion and proliferation; however, this helps them maintain surface smoothness and helps the disc's movement [27].
The effects of addition of TiO2 nanofilms, as a superhydrophilic surface modification to a scaffold fabricated from the aforementioned polymers, have been inspected [4,39].An increase in thickness of TiO2 nanolayer was contributed to enhanced cell proliferation and protein synthesis, and same as the native TMJ disc collagen type 1 surpassed the type 2 [4].The scaffold also proved to be biocompatible and cell adherent [4].

Poly (glycerol sebacate)
Poly (glycerol sebacate) is a biocompatible and biodegradable polymer [40].Poly (glycerol sebacate) scaffold along with fibro-chondrocytes has shown random distribution of collagen and GAG.Higher fibro-chondrocytes seeding density results in higher cellular content, and EMC production [40].Higher fibro-chondrocytes seeding density also results in a higher collagen and GAG content after 4 weeks [40].

Decellularized tissues
Decellularized ECM scaffolds are considered costeffective method for tissue regeneration, since they can maintain bioactive molecules such as growth factors [41].Decellularized scaffolds derived from native TMJ disc are believed to be biocompatible, bioactive and maintain chemical and mechanical properties of the tissues they were derived from [1,[42][43].It is believed that decellularization changes the ECM architecture with compacting ECM fiber, which consequently increases the compressive modulus to an extreme extent, decreases permeability, and causes a non-uniform cell distribution [1,10].There is also evidence of increased collagen and decreased GAG content [1].CO2 laser is suggested to be helpful for managing the undesired alterations by producing organized microporosity in the scaffold, which improves permeability, correction of fibril structures, which results in a compressive modulus close to that of native disc and finally uniform cell distribution, which is probably attributed to the increase in elastic modulus [10].The last one helps the scaffold degradation to be post-pones [10].There is a debate that presence of abundant dense fibrils in a decellularized scaffold might limit cell proliferation [44].Previous studies suggest that preparation of decellularized tissue in form of hydrogel overcome this challenge due to loose structure and fast degradation [1,10].The downside is that fast degradation may not provide proper stability during the whole regeneration process [45].The condition of decellularized hydrogel preparation can affect its mechanical properties [1].It is argued that an increase in s-hear rate, temperature, and concentration of decellularized ECM decreases viscosity, increases storage modulus and increases compressive strength, respectively [1].
The greatest challenge right now might be finding an appropriate surgical approach for implementation of the scaffolds.Besides, proper clinical guidelines should be developed.We have come a long way but there is still a lot to do before tissue engineering becomes a routine clinical option.
This narrative review preformed a search with both strengths and limitations.The use of two major databases, PubMed and Google Scholar, increased the probability of finding relevant studies.However, the search only included studies published within the last 10 years.
Furthermore, the inclusion of only specific types of studies may limit the range of evidence.

Conclusion
Scaffolds are an essential part of TMJ disc regeneration.
Natural biomaterials, synthetic biomaterials, and decellularized tissues are commonly used as scaffolds for this purpose.Although synthetic scaffolds offer more control over structural properties, natural scaffolds, such as collagen, hyaluronic acid, chitosan and alginate are still widely used due to their ability to promote cell attachment, signaling, and proliferation.The type of scaffold significantly affects the mechanical, structural, and biological properties of the regenerated tissue.Therefore, the development of an ideal scaffold that mimics the native TMJ disc is of great importance in the field of tissue engineering.
Although there is not a commercial product on the market yet, several in vivo studies have managed to prepare appropriate scaffolds for regeneration of TMJ disc.
However, longitudinal studies are required to observe and evaluate the effect of these scaffolds in a long run.

Figure 1 :
Figure 1: Tissue engineering for TMJ disc regeneration major part of ECM is made up of collagen fibers (mostly type 1) which mainly have circumferential orientation in the peripheral area and anteroposterior orientation in the intermediate area, and the rest is composed of elastic fibers and glycosaminoglycans (GAG).GAGs are main-

Table 1 :
Scaffolds used for TMJ disc regeneration in in vitro studies